EP1305422B1 - Permeases 2,5-dkg - Google Patents

Permeases 2,5-dkg Download PDF

Info

Publication number
EP1305422B1
EP1305422B1 EP01959520A EP01959520A EP1305422B1 EP 1305422 B1 EP1305422 B1 EP 1305422B1 EP 01959520 A EP01959520 A EP 01959520A EP 01959520 A EP01959520 A EP 01959520A EP 1305422 B1 EP1305422 B1 EP 1305422B1
Authority
EP
European Patent Office
Prior art keywords
dkg
nucleic acid
seq
polypeptide
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP01959520A
Other languages
German (de)
English (en)
Other versions
EP1305422A2 (fr
Inventor
Veronique A. Dartois
James A. Hoch
Fernando Valle
Manoj Kumar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MicroGenomics Inc
Danisco US Inc
Original Assignee
MicroGenomics Inc
Danisco US Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MicroGenomics Inc, Danisco US Inc filed Critical MicroGenomics Inc
Publication of EP1305422A2 publication Critical patent/EP1305422A2/fr
Application granted granted Critical
Publication of EP1305422B1 publication Critical patent/EP1305422B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/58—Aldonic, ketoaldonic or saccharic acids
    • C12P7/60—2-Ketogulonic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/26—Klebsiella (G)

Definitions

  • the present invention relates generally to microbial transporter proteins and, more specifically, to novel 2,5-diketo-D-gluconic acid (2,5-DKG) permeases.
  • Figure 2 shows schematically the enzymatic reactions that take place in the bioconversion of D-glucose to 2-KLG.
  • the enzymatic reactions that lead from D-glucose, to D-gluconic acid, to 2-keto-D-gluconic acid (2-KDG), to 2,5-diketo-D-gluconic acid (2,5-DKG) take place at the surface of the bacterial cell. 2,5-DKG must then enter the cell in order for its enzymatic conversion to 2-KLG.
  • the invention provides an isolated nucleic acid molecule as defined in the claims encoding a polypeptide which has 2,5-DKG permease activity.
  • the isolated nucleic acid molecule contains a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9.
  • the isolated nucleic acid molecule contains a nucleotide sequence which encodes a polypeptide having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, and 10.
  • vectors and cells containing isolated nucleic acid molecules as defined in the claims encoding polypeptides having 2,5-KDG permease activity are also provided.
  • the cells are bacterial cells selected from the genera Pantoea and Klebsiella .
  • the invention also provides methods of identifying and isolating nucleic acid molecules encoding polypeptides which have 2,5-DKG permease activity. Also provided are methods of enhancing 2-KLG production, by expressing the nucleic acid molecules of the invention in suitable bacterial cells.
  • isolated polypeptides as defined in the claims having 2,5-DKG permease activity.
  • the invention also provides antibodies specific for such polypeptides.
  • the invention provides novel nucleic acid molecules as defined in the claims encoding polypeptides having 2,5-DKG permease activity, and related products and methods.
  • the molecules of the invention can advantageously be used to increase the efficiency of 2-KLG bioproduction, and thus to lower the cost of commercial ascorbic acid production.
  • Naturally occurring 2,5-DKG permeases are polypeptides localized to the cytoplasmic membrane of microorganisms, which are predicted, using commercially available topology prediction programs, to contain about 10 to 12 transmembrane domains.
  • Each transmembrane spanning segment is about 20 amino acids in length, with the intracellular and extracellular loops ranging from about 2 to about 83 amino acids in length.
  • the loop between the fifth and sixth transmembrane domain spanning segments is larger than the other loops.
  • Naturally occurring 2,5-DKG permeases are typically about 350-550 amino acids in length, such as about 400-450 amino acids in length, and particularly about 425-440 amino acids in length.
  • SEQ ID NO:1 PE1 from an environmental source
  • SEQ ID NO:3 PE6 from an environmental source
  • SEQ ID NO:5 PK1 from Klebsiella oxytoca
  • SEQ ID NO:7 PrmA from Pantoea citrea
  • SEQ ID NO:9 PrmB from Pantoea citrea
  • comparative SEQ ID NO:11 YiaX2 from Klebsiella oxytoca
  • SEQ ID NO:2 The corresponding encoded 2,5-DKG permease amino acid sequences are set forth as SEQ ID NO:2 (PE1); SEQ ID NO:4 (PE6); SEQ ID NO:6 (PK1); SEQ ID NO:8 (prmA); SEQ ID NO:10 (prmB); and comparative SEQ ID NO:12 (YiaX2).
  • 2,5-DKG permeases from different microorganisms exhibit extensive amino acid sequence relatedness over their entire length, as is evidenced by the six-way sequence alignment shown in Figure 1 .
  • the overall identity of the six permeases shown in Figure 1 is about 17%, and the overall similarity, taking into account conservative substitutions, is about 43%.
  • 2,5-DKG permeases disclosed herein can be further subdivided into two structural families.
  • the three 2,5-DKG permeases designated YiaX2, PE6 and PrmA are representative of one family of permeases, sharing about 50% overall identity in a three-way amino acid sequence alignment (YiaX2 is included for comparative purposes only).
  • the three 2,5-DKG permeases designated PK1, PE1 and PrmB are representative of a second family of related permeases, sharing about 60% overall identity in a three-way amino acid sequence alignment.
  • 2,5-DKG permease activity refers to the ability of the polypeptide, when expressed in its native orientation at the cell membrane, to transport 2,5-DKG across the cytoplasmic membrane, in comparison with an unrelated control polypeptide. Such transport can be either unidirectional or bidirectional.
  • 2,5-DKG permease activity can be determined by a variety of methods.
  • 2,5-DKG permease activity can be determined using a metabolic selection assay, as described further in the Example, below. Briefly, a bacterial cell either naturally deficient in 2,5-DKG permease activity, or made deficient in 2,5-DKG permease activity, is identified or produced.
  • bacterial cells can be made deficient in endogenous 2,5-DKG permease activity by preparing a deletion mutant of one or more endogenous 2,5-DKG permease genes, using the polymerase chain reaction, following methods known in the art.
  • the term "deficient,” as used in relation to a cell deficient in 2,5-DKG permease activity, is intended to refer to endogenous 2,5-DKG permease activity that is comparable to, or less than, the endogenous permease activity of a K. oxytoca strain deleted in the yiaX2 gene, such as the strain K. oxytoca ⁇ yiaX2 [ tkr idnO ], as assessed either by a growth assay or by a 2,5-DKG uptake assay.
  • a cell useful in a metabolic selection assay to determine 2,5-DKG permease activity of an expressed polypeptide can further naturally be capable of converting intracellular 2,5-DKG to carbon and energy, or made capable of such conversion by recombinant expression of appropriate metabolic enzymes.
  • a combination of nucleic acid molecules encoding a 2-keto-reductase ( tkr ) and a 5-keto-reductase ( idnO ), from any bacterial species can be expressed in the cell, which together provide the cell with the ability to catalyze the reduction of 2,5-DKG to gluconic acid. Gluconic acid can then be used by the cell as a carbon and energy source that supports cell growth.
  • An exemplary bacterial cell suitable for metabolic assays to determine 2,5-DKG permease activity is the strain K. oxytoca ⁇ yiaX2 [ tkr idnO ] shown in Figure 3 and described in the Example, below.
  • This strain has a deleted yiaX2 2,5-DKG permease gene, and also recombinantly expresses the tkr / idnD / idnO operon set forth as SEQ ID NO:13 on a high copy number plasmid.
  • nucleotides 292-1236 encode a 2-keto-reductase (tkr) (SEQ ID NO:14); nucleotides 1252-2280 encode an idonic acid dehydrogenase (idnD) (SEQ ID NO:15); and nucleotides 2293-3045 encode a 5-keto-reductase (idnO) (SEQ ID NO:16).
  • nucleic acid molecules encoding polypeptides which contain modifications from the amino acid,sequences designated SEQ ID NO:14 or 16, but which retain 2-keto-reductase activity or 5-keto-reductase activity, respectively can be used in metabolic assays.
  • Exemplary amino acid sequences have at least 60%, such as at least 70%, preferably 80%, 90%, 95% or greater identity to SEQ ID NOS:14 or 16, respectively.
  • the ability of such a bacterial cell to grow on medium containing 2,5-DKG as the sole carbon source, upon expression of a candidate 2,5-DKG permease, is a measure of the ability of the expressed permease to transport 2,5-DKG into the cell, and is thus a measure of its 2,5-DKG permease activity.
  • Each of SEQ ID NOS:2, 4, 6, 8, 10 and comparative 12 was demonstrated to have 2,5-DKG permease activity, as evidenced by the ability of K. oxytoca ⁇ yiaX2 [ tkr idnO ] expressing each permease to grow on 2,5-DKG as the sole carbon source.
  • 2,5-DKG permease activity can be determined by measuring uptake of labeled or unlabeled 2,5-DKG.
  • 2,5-DKG can be detectably labeled, such as with a fluorescent or radioactive tag.
  • the ability of a cell or membrane vesicle expressing a 2,5-DKG permease to take up the detectable label when provided with detectably labeled 2,5-DKG can be determined using detection assays specific for the particular label, which are well known in the art.
  • uptake of unlabeled 2,5-DKG can be measured by HPLC or other sensitive detection assay known in the art. Uptake of 2,5-DKG is thus a measure of permease activity.
  • Each of SEQ ID NOS:2, 4, 6, 8, 10 and comparative 12 exhibits 2,5-DKG permease activity as determined by assay of uptake of radiolabeled 2,5-DKG by bacterial cells expressing the recombinant permeases.
  • 2,5-DKG permease activity can be measured in any cell in which 2,5-DKG can be converted to a product, by measuring production of the product in the presence of extracellular 2,5-DKG.
  • a 2,5-DKG reductase intracellular 2,5-DKG is converted to 2-KLG.
  • the ability of the bacterial cell to produce 2-KLG when provided with extracellular 2,5-DKG, upon expression of a 2,5-DKG permease is a measure of the ability of the expressed permease to transport 2,5-DKG into the cell, and is thus a measure of its 2,5-DKG permease activity.
  • Intracellular 2-KLG can be detected, for example, using HPLC or other sensitive detection methods known in the art. Other metabolic products of 2,5-DKG can also be detected, by similar methods.
  • 2,5-DKG permease activity For instance, the change in pH across a cell or vesicle membrane as 2,5-DKG, an acid, is transported across the membrane can be detected. Similarly, a decrease over time in extracellular 2,5-DKG can be determined.
  • a 2,5-DKG permease of the invention can selectively transport 2,5-DKG.
  • selective refers to preferential transport of 2,5-DKG rather than 2-KLG into or out of the cell.
  • a permease that selectively transports 2,5-DKG will transport 2,5-DKG at least 2-fold, such as at least 5-fold, including greater than 10-fold more efficiently than it transports 2-KLG.
  • a permease that selectively transports 2,5-DKG is particularly advantageous in applications where it is desirable to increase intracellular production of 2-KLG, such as in the commercial production of ascorbic acid.
  • employing a permease that selectively transports 2,5-DKG prevents intracellular 2-KLG from competing with extracellular 2,5-DKG for permease-mediated transport through the membrane, and increases the overall efficiency of intracellular 2-KLG production.
  • a metabolic assay can be designed in which a bacterial cell can convert either intracellular 2,5-DKG or 2-KLG to carbon and energy.
  • the relative ability of the cell to grow on 2,5-DKG as the sole carbon source, compared with its ability to grow on 2-KLG as the sole carbon source is a measure of the ability of the expressed permease to selectively transport 2,5-DKG.
  • the invention provides an isolated nucleic acid molecule as defined in the claims encoding a polypeptide which has 2,5-DKG permease activity.
  • the nucleic acid molecules of the invention are suitable for a variety of commercial and research applications.
  • one or more of the invention nucleic acid molecules can be expressed in bacterial cells in order to enhance the rate of uptake of 2,5-DKG by the cells.
  • Enhancing uptake of 2,5-DKG has a variety of applications, such as in commercial production of 2,5-DKG itself, or commercial production of any metabolic product of 2,5-DKG.
  • 2,5-DKG uptake is a rate limiting step in the biosynthesis of 2-KLG, which is a stable intermediate in the synthesis of ascorbic acid.
  • 2-KLG can thus be obtained from bacterial cells expressing 2,5-DKG permeases, and converted to ascorbic acid.
  • nucleic acid molecules can be used as probes or to design primers to identify and isolate 2,5-DKG permease homologs from additional species, or as templates for the production of mutant permeases, using methods known in the art and described further below.
  • permeases can have advantageous properties compared with the 2,5-DKG permeases disclosed herein as SEQ ID NOS:2, 4, 6, 8, and 10, such as greater enzymatic activity or greater 2,5-DKG selectivity.
  • an isolated nucleic acid molecule can be a molecule operatively linked to an exogenous nucleic acid sequence.
  • An isolated nucleic acid molecule can also be a molecule removed from some or all of its normal flanking nucleic acid sequences, such as removed from one or more other genes within the operon in which the nucleic acid molecule is normally found.
  • isolated is intended to mean that the nucleic acid molecule does not contain any of the flanking open reading frames (orfs) present in the K. oxytoca yia operon, such as the orfs designated lyxK and orf1, described in WO 00/22170 .
  • An isolated molecule can alternatively, or additionally, be a "substantially pure" molecule, in that the molecule is at least 60%, 70%, 80%, 90 or 95% free from cellular components with which it is naturally associated.
  • An isolated nucleic acid molecule can be in any form, such as in a buffered solution, a suspension, a heterologous cell, a lyophilized powder, or attached to a solid support.
  • nucleic acid molecule refers to a polynucleotide of natural or synthetic origin.
  • a nucleic acid molecule can be single- or double-stranded genomic DNA, cDNA or RNA, and represent either the sense or antisense strand or both.
  • a nucleic acid molecule can thus correspond to the recited sequence, to its complement, or both.
  • nucleic acid molecule is intended to include nucleic acid molecules that contain one or more non-natural nucleotides, such as nucleotides having modifications to the base, the sugar, or the phosphate portion, or having one or more non-natural linkages, such as phosphothioate linkages. Such modifications can be advantageous in increasing the stability of the nucleic acid molecule, particularly when used in hybridization applications.
  • nucleic acid molecule is intended to include nucleic acid molecules modified to contain a detectable moiety, such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin. Nucleic acid molecules containing such moieties are useful as probes for detecting the presence or expression of a 2,5-DKG permease nucleic acid molecule.
  • a detectable moiety such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin.
  • the isolated nucleic acid molecule encoding a polypeptide which has 2,5-DKG permease activity contains a nucleotide sequence having at least 80% identity to any of the 2,5-DKG permease nucleic acid molecules designated SEQ ID NOS:1, 3, 5, 7, or 9.
  • SEQ ID NOS:1, 3, 5, 7, or 9 a nucleotide sequence having at least 80% identity to any of the 2,5-DKG permease nucleic acid molecules designated SEQ ID NOS:1, 3, 5, 7, or 9.
  • such a molecule will have at least 90% identity to any of these recited SEQ ID NOS, such as 95%, 98%, 99% or greater identity to SEQ ID NOS:1, 3, 5, 7, or 9.
  • the isolated nucleic acid molecule encoding a polypeptide which has 2,5-DKG permease activity contains a nucleotide sequence which encodes a polypeptide having at least 80% identity to any of the 2,5-DKG permease polypeptides designated SEQ ID NOS:2, 4, 6, 8, or 10.
  • the encoded polypeptide will have at least 90% identity to any of 95%, 98%, 99% or greater identity to SEQ ID NOS:2, 4, 6, 8, or 10.
  • percent identity with respect to a nucleic acid molecule or polypeptide of the invention is intended to refer to the number of identical nucleotide or amino acid residues between the aligned portions of two sequences, expressed as a percent of the total number of aligned residues, as determined by comparing the entire sequences using a CLUSTAL V computer alignment and default parameters.
  • CLUSTAL V alignments are described in Higgens, Methods Mol. Biol. 25:307-318 (1994 ), and an exemplary CLUSTAL V alignment of 2,5-DKG permease amino acid sequences is presented in Figure 1 .
  • nucleotide sequence of a native nucleic acid molecule can be modified and still encode an identical or substantially similar polypeptide.
  • degenerate variants of SEQ ID NOS:1, 3, 5, 7, or 9 are exemplary invention nucleic acid molecules encoding polypeptides having 2,5-DKG permease activity.
  • nucleic acid molecules encoding 2,5-DKG permeases from other species of microorganisms are exemplary invention nucleic acid molecules.
  • YiaX2 is included for comparative purposes only.
  • the two most similar of the disclosed 2,5-DKG permease nucleotide sequences, SEQ ID NO:5 (PK1) and SEQ ID NO:1 (PE1) share 86% identity across their length.
  • the six permeases disclosed herein also share substantial amino acid sequence identity over their entire length, as described previously.
  • PK1 from Klebsiella oxytoca SEQ ID NO:6
  • PE1 from an environmental source SEQ ID NO:2
  • SEQ ID NO:2 The amino acid sequence in the GenBank database most closely related to a 2,5-DKG permease, which is a putative tartrate transporter from Agrobacterium vitis (GenBank Accession U32375 or U25634) is 33% identical to comparative SEQ ID NO:12 (YiaX2), and shares less identity with the other disclosed 2,5-DKG permeases.
  • sequences with some degree of identity in the GenBank database to the disclosed 2,5-DKG permease include membrane transporter proteins from a variety of species, including phthalate transporter proteins from B. cepacia (AF152094) and P. putida (D13229); hydroxyphenylacetate transporters from S. dublin (AF144422) and E. coli (Z37980); and probable transporter proteins from S. coelicolor (AL136503 and AL132991) each of which has about 27% or less identity at the amino acid level to the recited SEQ ID NOS.
  • membrane transporter proteins from a variety of species including phthalate transporter proteins from B. cepacia (AF152094) and P. putida (D13229); hydroxyphenylacetate transporters from S. dublin (AF144422) and E. coli (Z37980); and probable transporter proteins from S. coelicolor (AL136503 and AL132991) each of which has about 27% or less identity at the amino acid level
  • nucleic acid molecules as defined in the claims include nucleic acid molecules that encode polypeptides having 2,5-DKG permease activity from any microbial species.
  • Microorganisms that contain 2,5-DKG permeases can be recognized by their ability to actively transport 2,5-DKG, such that they can grow on 2,5-DKG as the sole carbon source, or incorporate 2,5-DKG in an uptake assay.
  • Such microorganisms can include, for example, bacteria, including Archaebacteria, gram positive and gram negative bacteria; yeast; and fungi.
  • Exemplary bacteria which contain 2,5-DKG permeases include Proteobacteria, and more specifically Enterobacteria and Pseudomonads (e.g. P. aeruginosa), as described in the Example.
  • Exemplary Enterobacteria include species from the genera Klebsiella (e.g. K. oxytoca, from which SEQ ID NOS:5 and 11 were obtained) and Pantoea (e.g. P. citrea , from which SEQ ID NOS:7 and 9 were obtained, and P. agglomerans ).
  • Sources of such microorganisms include public repositories, such as the American Type Culture Collection (ATCC), and commercial sources.
  • ATCC American Type Culture Collection
  • Klebsiella oxytoca e.g. ATCC 13182
  • Aerobacter aerogenes e.g. Aerobacter aerogenes
  • Klebsiella aerogenes e.g. Aerobacter aerogenes
  • Klebsiella aerogenes e.g. Aerobacter aerogenes
  • Klebsiella aerogenes e.g. Aerobacter aerogenes
  • Klebsiella aerogenes e.g. ATCC 2
  • Pantoea agglomerans e.g. ATCC 21998) has alternatively been described as Erwinia herbicola and Acetomonas albosesamae.
  • the terms "Klebsiella” and "Pantoea,” as used herein, are intended to refer to the genera of the strains deposited as ATCC 13182 and 21998, respectively.
  • microorganisms from which 2,5-DKG permease nucleic acid molecules can be obtained are microorganisms present in environmental samples.
  • the 2,5-DKG permease nucleic acid molecules designated SEQ ID NOS:1 and 3 were obtained from environmental samples.
  • the term "environmental sample” refers to a sample obtained from natural or man-made environments, which generally contains a mixture of microorganisms.
  • Exemplary environmental samples are samples of soil, sand, freshwater or freshwater sediments, marine water or marine water sediments, industrial effluents, hot springs, thermal vents, and the like. Within an environmental sample there are likely to be microorganisms that are unidentified, and also microorganisms that are uncultivable. Isolation of 2,5-DKG permease molecules as defined in the claims from microorganisms present in environmental samples does not require either identification or culturing of the microorganism.
  • nucleic acid molecules of the invention include nucleic acid molecules encoding amino acid sequences that are modified by one or more amino acid additions, deletions or substitutions with respect to the native sequence of SEQ ID NOS:2, 4, 6, 8, or 10. Such modifications can be advantageous, for example, in enhancing the stability, expression level, enzymatic activity, or 2,5-DKG selectivity of the permease. If desired, such modifications can be randomly generated, such as by chemical mutagenesis, or directed, such as by site-directed mutagenesis of a native permease sequence, using methods well known in the art.
  • An amino acid sequence that is modified from a native permease amino acid sequence can include one or more conservative amino acid substitutions, such as substitution of an apolar amino acid with another apolar amino acid (such as replacement of leucine with an isoleucine, valine, alanine, proline, tryptophan, phenylalanine or methionine); substitution of a charged amino acid with a similarly charged amino acid (such as replacement of a glutamic acid with an aspartic acid, or replacement of an arginine with a lysine or histidine); or substitution of an uncharged polar amino acid with another uncharged polar amino acid (such as replacement of a serine with a glycine, threonine, tyrosine, cysteine, asparagine or glutamine).
  • a modified amino acid sequence can also include one or more nonconservative substitutions without adversely affecting the desired biological activity.
  • Alignment of the six 2,5-DKG permeases described herein also provides guidance as to regions where additions and deletions are likely to be tolerated.
  • the N and C termini, and the region around amino acids 225-250 appear to be regions that are relatively tolerant of amino acid insertions and deletions, as evidenced by gaps in the sequence alignment.
  • Modified 2,5-DKG permeases can thus include "tag" sequences at such sites, such as epitope tags, histidine tags, glutathione-S-transferase (GST) and the like, or sorting sequences.
  • additional sequences can be used, for example, to facilitate purification or characterization of a recombinant 2,5-DKG permease.
  • nucleic acid molecule is a nucleic acid molecule of the invention
  • confirmation that any particular nucleic acid molecule is a nucleic acid molecule of the invention can be obtained by determining the 2,5-DKG permease activity of the encoded polypeptide, using one or more of the functional assays described herein.
  • the invention further provides an isolated nucleic acid molecules as defined in the claims encoding a polypeptide which has 2,5-DKG permease activity, wherein the nucleic acid molecule is operatively linked to a promoter of gene expression.
  • operatively linked is intended to mean that the nucleic acid molecule is positioned with respect to either the endogenous promoter, or a heterologous promoter, in such a manner that the promoter will direct the transcription of RNA using the nucleic acid molecule as a template.
  • Methods for operatively linking a nucleic acid to a desired promoter include, for example, cloning the nucleic acid into a vector containing the desired promoter, or appending the promoter to a nucleic acid sequence using PCR.
  • a nucleic acid molecule operatively linked to a promoter of RNA transcription can be used to express 2,5-DKG transcripts and polypeptides in a desired host cell or in vitro transcription-translation system.
  • promoter to operatively link to an invention nucleic acid molecule will depend on the intended application, and can be determined by those skilled in the art. For example, if a particular gene product may be detrimental to a particular host cell, it may be desirable to link the invention nucleic acid molecule to a regulated promoter, such that gene expression can be turned on or off.
  • An exemplary inducible promoter known in the art is the lacPO promoter/operator, which is repressed by the lacI q gene product provided by certain host cells, and induced in the presence of 0.01 to 1 mM IPTG (see Example, below). For other applications, weak or strong constitutive promoters may be preferred.
  • the invention further provides a vector containing an isolated nucleic acid molecule as defined in the claims encoding a polypeptide which has 2,5-DKG permease activity.
  • the vectors of the invention will generally contain elements such as a bacterial origin of replication, one or more selectable markers, and one or more multiple cloning sites.
  • the choice of particular elements to include in a vector will depend on factors such as the intended host cell or cells; whether expression of the inserted sequence is desired; the desired copy number of the vector; the desired selection system, and the like. The factors involved in ensuring compatibility between a host and a vector for different applications are well known in the art.
  • the isolated nucleic acid molecules will generally be operatively linked to a promoter of gene expression, as described above, which may be present in the vector or in the inserted nucleic acid molecule. In cloning and subcloning applications, however, promoter elements need not be present.
  • An exemplary vector suitable for both cloning applications and for expressing 2,5-DKG permeases in different bacterial species is the low copy number plasmid pCL1920 described by Lerner et al., Nucleic Acids Res. 18:4621 (1994 ), which contains a spectinomycin resistance gene (see Example, below).
  • cells containing an isolated nucleic acid molecule as defined in the claims encoding a polypeptide which has 2,5-DKG permease activity will generally be contained within a vector compatible with replication in the particular host cell. However, for certain applications, incorporation of the nucleic acid molecule into the bacterial genome will be preferable.
  • the cells of the invention can be any cells in which a 2,5-DKG permease will be expressed and folded into an active conformation.
  • Guidance in choosing appropriate host cells is provided by identifying cell types which express other functional 10 to 12 transmembrane transporter proteins.
  • 10 to 12 transmembrane transporter proteins are found in a variety of bacterial species, as well as in yeast (e.g. S. pombe ) , Arabidopsis, and Drosophila. Therefore, depending on the particular application for the host cell, a host cell of the invention can be a bacterial cell, yeast, Arabidopsis or Drosophila cell.
  • the cell is a bacterial cell.
  • the choice of bacterial cell will depend on the intended application.
  • the cell can be any convenient laboratory strain of bacteria, such as E. coli, which can be transformed with the isolated nucleic acid molecules and vectors of the invention by methods well known in the art.
  • the cell can be a bacterial strain suitable for metabolic assays, such as a strain which endogenously expresses, or which is engineered to express, enzymes that catalyze the conversion of 2,5-DKG to essential products.
  • An exemplary strain suitable for metabolic assays is the K. oxytoca ⁇ yiaX2 [ tkr idnO ] strain designated MGK002[pDF33] described further in the Example, below, which provides for the conversion of intracellular 2,5-DKG to gluconic acid, which can be used as a carbon and energy source.
  • the cell can be a bacterial strain which endogenously expresses, or which is engineered to express, a 2,5-DKG reductase.
  • 2,5-DKG reductases are found in genera including Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus, Pseudomonas, Bacillus, Citrobacter and Corynebacterium. Therefore, a cell of the invention can be a bacterial cell of any of these genera, or a bacterial cell engineered to express a 2,5-DKG reductase of any of these genera.
  • a cell able to produce 2,5-DKG metabolites will preferably also be able to catalyze the extracellular production of 2,5-DKG from an inexpensive carbon source, such as glucose.
  • An exemplary pathway from D-glucose to 2,5-DKG involves the enzymatic conversion of D-glucose to D-gluconic acid (catalyzed by D-glucose dehydrogenase), from D-gluconic acid to 2-Keto-D-gluconic acid (catalyzed by D-gluconate dehydrogenase), and from 2-Keto-D-gluconic acid to 2,5-DKG (catalyzed by 2-Keto-D-gluconic acid dehydrogenase), as is shown in Figure 2 .
  • These steps can be carried out by organisms of several genera, including Gluconobacter, Acetobacter and Erwinia (also called Pantoea).
  • a bacterial cell useful for the production of 2-KLG from D-glucose is the Pantoea aggolmerans (also referred to as Erwinia herbicola or Acetomonas albosesamae ) strain described in U.S. Patent No. 5,032,514 , designated ATCC 21998 ptrp 1-35 tkrA ⁇ 3, or a derivative of this strain with improved properties.
  • Contemplated improvements to this strain which can be produced by genetic engineering, include deletion of enzymes that divert glucose to metabolites other than 2-KLG, such that yield of 2-KLG is increased.
  • Other contemplated improvements to this strain include mutations that provide for improved recovery and purification of 2-KLG.
  • the Pantoea strain described in U.S. Patent No. 5,032,514 recombinantly expresses a 2,5-DKG reductase from Corynebacterium (described in U.S. Patent No. 4,757,012 ).
  • the strain further contains a mutation that results in a non-functional tkrA gene and is thus deficient in 2-keto reductase activity. Mutation of the tkrA gene is advantageous in reducing metabolic diversion of 2-KLG to L-idonic acid, and metabolic diversion of 2,5-DKG to 5-keto-D-gluconate from 2-KLG.
  • the cells of the invention can contain one, two or more isolated nucleic acid molecules as defined by the claims that encode polypeptides having 2,5-DKG permease activity.
  • the cell can contain an isolated nucleic acid molecule encoding at least one polypeptide having at least 80% identity to any of SEQ ID NOS:2, 4, 6, 8, or 10, and optionally will contain two or more such nucleic acid molecules, in any combination.
  • at least one such encoded polypeptide selectively transports 2,5-DKG.
  • a bacterial cell of the invention suitable for bioproduction of 2-KLG contains an isolated nucleic acid molecule encoding a polypeptide having at least 95% identity to the 2,5-DKG selective permease designated SEQ ID NO:8 (prmA); and optionally further containing at least one isolated nucleic acid molecule encoding a polypeptide having at least 95% identity to a 2,5-DKG permease selected from the group consisting of SEQ ID NO:4 (PE6), SEQ ID NO:10 (prmB) and SEQ ID NO:6 (PK1).
  • the invention also provides a method of enhancing production of 2-KLG.
  • the method consists of culturing a bacterial cell, wherein the cell contains an isolated nucleic acid molecule as defined by the claims encoding a polypeptide which has 2,5-DKG permease activity, under conditions wherein the encoded 2,5-DKG permease is expressed and intracellular 2,5-DKG is converted to 2-KLG.
  • Cells suitable for this purpose such as the Pantoea strain described in U.S. Patent No. 5,032,514 , have been described above.
  • the 2-KLG so produced can be chemically or enzymatically converted to a desired product such as ascorbic acid, following methods known in the art.
  • nucleic acid molecules as defined in the claims that specifically hybridize to a nucleic acid molecule encoding a 2,5-DKG permease.
  • the term “specifically hybridize” refers to the ability of a nucleic acid molecule to hybridize, under stringent hybridization conditions as described below, to a nucleic acid molecule that encodes a 2,5-DKG permease, without hybridizing to a substantial extent under the same conditions with nucleic acid molecules that do not encode 2,5-DKG permeases, such as unrelated molecules that fortuitously contain short regions of identity with a permease sequence.
  • nucleic acid molecule that "specifically hybridizes" is of a sufficient length and contains sufficient distinguishing sequence from a 2,5-DKG permease to be characteristic of the 2,5-DKG permease.
  • Such a molecule will generally hybridize, under stringent conditions, as a single band on a Northern blot or Southern blot prepared from mRNA of a single species.
  • stringent conditions refers to conditions equivalent to hybridization of a filter-bound nucleic acid molecule to a nucleic acid in a solution containing 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing the filter in 0.1X SSPE, and 0.1% SDS at 65°C twice for 30 minutes.
  • Equivalent conditions to the stringent conditions set forth above are well known in the art, and are described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992 ).
  • Nucleotide sequences that are characteristic of each of SEQ ID NOS:1, 3, 5, 7 or 9, or which are common to two, three or more of SEQ ID NOS:1, 3, 5, 7, 9 or 11 can readily be determined by aligning the sequences using a CLUSTAL V alignment program. Oligonucleotides containing regions which are common to two or more different 2,5-DKG permease nucleic acid molecules can advantageously be used as PCR primers or hybridization probes to isolate or detect nucleic acid molecules encoding 2,5-DKG permeases from other species.
  • nucleic acid molecules as defined in the claims which encode polypeptides having 2,5-DKG permease activity, will be subsequently referred as "2,5-DKG permease nucleic acid molecules.”
  • the isolated 2,5-DKG permease nucleic acid molecules as defined in the claims can be prepared by methods known in the art. The method chosen will depend on factors such as the type and size of nucleic acid molecule one intends to isolate; whether or not it encodes a biologically active polypeptide (e.g. a polypeptide having permease activity); and the source of the nucleic acid molecule. Those skilled in the art can isolate or prepare 2,5-DKG permease nucleic acid molecules as genomic DNA or desired fragments therefrom; as full-length cDNA or desired fragments therefrom; or as full-length mRNA or desired fragments therefrom, from any microorganism of interest.
  • a biologically active polypeptide e.g. a polypeptide having permease activity
  • An exemplary method of preparing a 2,5-DKG permease nucleic acid molecule is by isolating a recombinant construct which encodes and expresses a polypeptide having 2,5-DKG permease activity.
  • one useful method is to provide a metabolic selection system where bacterial cell growth is made dependent on expression of a 2,5-DKG permease, introducing expressible DNA, such as a cDNA or genomic library, into the assay cells, selecting surviving cells under the selective conditions, and isolating the introduced DNA.
  • a screening method can be designed, such that a cell will exhibit a detectable signal only when expressing a functional 2,5-DKG permease.
  • An exemplary detectable signal is intracellular incorporation of a detectable label present on 2,5-DKG. Additionaly screening and selection strategies suitable for identifying nucleic acid molecules encoding metabolic enzymes are described, for example, in PCT publication WO 00/22170 and U.S. Patent Nos. 5,958,672 and 5,783,431 .
  • a further method for producing an isolated 2,5-DKG permease nucleic acid molecule involves amplification of the nucleic acid molecule using 2,5-DKG permease-specific primers and the polymerase chain reaction (PCR). Using PCR, a 2,5-DKG permease nucleic acid molecule having any desired boundaries can be amplified exponentially starting from as little as a single gene or mRNA copy, from any cell having a 2,5-DKG permease gene.
  • PCR polymerase chain reaction
  • primers for isolating additional 2,5-DKG permease nucleic acid molecules.
  • Such primers are preferably degenerate oligonucleotides that encode, or are complementary to, short consensus amino acid sequences present in two or more of the 2,5-DKG permeases disclosed herein, such as oligonucleotides that encode 10 or more contiguous amino acids present in at least two of SEQ ID NOS: 2, 6 and 10, or oligonucleotides that encode 10 or more contiguous amino acids present in at least two of SEQ ID NOS:4, 8 and 12 (SEQ ID NO. 12 is included for comparative purposes only).
  • Such sequences can be determined from an alignment of amino acid sequences shown in Figure 1 .
  • Exemplary amino acid sequences present in at least two of SEQ ID NOS:2, 6 and 10 are amino acids 19-31, 115-124, 146-156, and 339-348 of SEQ ID NO:2.
  • Exemplary amino acid sequences present in at least two of SEQ ID NOS:4, 8 and 12 are amino acids 55-64, 60-69, 252-261, and 370-379 of SEQ ID NO:8.
  • Methods are well known in the art to determine or modify PCR reaction conditions when using degenerate primers to isolate a desired nucleic acid molecule.
  • the amplified product can subsequently be sequenced, used as a hybridization probe, or used for 5' or 3' RACE to isolate flanking sequences, following procedures well known in the art and described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (2000 ).
  • an isolated 2,5-DKG permease nucleic acid molecule can be identified by screening a library, such as a genomic library, cDNA library or expression library, with a detectable nucleic acid molecule or antibody.
  • libraries are commercially available from a variety of microorganisms, or can be produced from any available microorganism or environmental sample of interest using methods described, for example, in PCT publication WO 00/22170 .
  • the library clones identified as containing 2,5-DKG permease nucleic acid molecules can be isolated, subcloned and sequenced by routine methods.
  • 2,5-DKG permease nucleic acid molecules can be produced by direct synthetic methods.
  • a single stranded nucleic acid molecule can be chemically synthesized in one piece, or in several pieces, by automated synthesis methods known in the art.
  • the complementary strand can likewise be synthesized in one or more pieces, and a double-stranded molecule made by annealing the complementary strands.
  • Direct synthesis is particularly advantageous for producing relatively short molecules, such as oligonucleotide probes and primers, and also for producing nucleic acid molecules containing modified nucleotides or linkages.
  • the invention also provides an isolated polypeptide as defined in the claims which has 2,5-DKG permease activity.
  • isolated polypeptides when expressed in their normal configuration at the cell membrane, are useful in applications in which enhanced uptake of 2,5-DKG is desirable, such as in bioproduction of 2-KLG.
  • the isolated polypeptides of the invention can also be added to a culture medium, preferably in a membrane vesicle, to compete with membrane-bound permeases for 2,5-DKG, and thus to stop 2,5-DKG uptake.
  • isolated polypeptides having 2,5-DKG permease activity can be used to regulate production of 2,5-DKG metabolites.
  • an "isolated" polypeptide of the invention is altered by the hand of man from how it is found in its natural environment.
  • an isolated 2,5-DKG permease can be a molecule that is recombinantly expressed, such that it is present at a higher level in its native host, or is present in a different host.
  • an "isolated" 2,5-DKG permease of the invention can be a substantially purified molecule.
  • Substantially purified 2,5-DKG permeases can be prepared by methods known in the art.
  • isolated is intended to mean that polypeptide is not present in association with the polypeptides expressed by other genes in the K. oxytoca yia operon, such as the genes designated lyxK and orf1 , described in WO 00/22170 .
  • an isolated polypeptide having 2,5-DKG permease activity contains an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, or 10.
  • the encoded polypeptide will have at least 90% identity to any of the recited SEQ ID NOS, including 95%, 98%, 99% or greater identity.
  • the 2,5-DKG polypeptide need not be in its native configuration to be recognized.
  • 2,5-DKG permease polypeptides of the invention have been described above with respect to the encoding nucleic acid molecules as defined in the claims, and are equally applicable in reference to the isolated polypeptides as defined in the claims. Isolated polypeptides having 2,5-DKG permease activity, will subsequently be referred to as "2,5-DKG permeases.”
  • 2,5-DKG permeases can alternatively be prepared by biochemical procedures, by isolating membranes from bacteria that naturally express, or recombinantly express, 2,5-DKG permeases.
  • the membranes can be further fractionated by size or affinity chromatography, electrophoresis, or immunoaffinity procedures, to achieve the desired degree of purity. Purification can be monitored by a variety of procedures, such as by immunoreactivity with 2,5-DKG permease antibodies, or by a functional assay.
  • 2,5-DKG permeases can be produced by chemical synthesis.
  • chemically synthesized molecules can include D-stereoisomers, non-naturally occurring amino acids, and amino acid analogs and mimetics.
  • detectably labeled amino acids such as radiolabeled or fluorescently labeled amino acids.
  • An isolated 2,5-DKG permease as defined in the claims can further be conjugated to carrier molecules, such as keyhole lympet hemocyanin, which can enhance recognition by the immune system of the isolated 2,5-DKG permease for production of antibodies.
  • carrier molecules such as keyhole lympet hemocyanin
  • the 2,5-DKG permease can be chemically or enzymatically derivatized, such as by acylation, phosphorylation or glycosylation.
  • the invention also provides an antibody specific for a polypeptide having 2,5-DKG permease activity, such as an antibody specific for a polypeptide having the amino acid sequence of any of SEQ ID NOS:2, 4, 6, 8, or 10.
  • the antibodies of the invention can be used, for example, to detect or isolate 2,5-DKG permeases from expression libraries or cells.
  • antibody is intended to include molecules having specific binding activity for a 2,5-DKG permease as defined in the claims of at least about 1 x 10 5 M -1 , preferably at least 1 x 10 7 M -1 , more preferably at least 1 x 10 9 M -1 .
  • the term “antibody” includes both polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies (e.g. Fab, F(ab') 2 , Fd and Fv fragments and the like).
  • the term “antibody” is intended to encompass non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, CDR-grafted antibodies and humanized antibodies, as well as antigen-binding fragments thereof.
  • Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains. Such methods are described, for example, in Huse et al. Science 246:1275-1281 (1989 ); Winter and Harris, Immunol.
  • This example shows the isolation and characterization of nucleic acid molecules encoding six novel polypeptides having 2,5-DKG permease activity.
  • WO 002170 describes the identification and sequencing of an operon from Klebsiella oxytoca, designated the yia operon, which contains eight putative open reading frames. Because disruption of this operon abolished the ability of K. oxytoca to utilize ascorbic acid as the sole carbon source, the yia operon was predicted to be involved in the catabolism of ascorbic acid. The functions of the polypeptides encoded by the individual open reading frames in the yia operon were not described in WO 002170 .
  • K. oxytoca was able to grow on 2,5-DKG as a sole carbon source and, therefore, it was concluded that K. oxytoca expressed a 2,5-DKG permease. It was predicted that such a permease would share structural properties with known bacterial transporter proteins, such as multiple transmembrane segments.
  • One of the uncharacterized open reading frames in the yia operon, designated yiaX2 encoded a transmembrane polypeptide with about 33% identity to a known tartrate transporter, and was thus considered a candidate 2,5-DKG permease.
  • M5a1 has also been described in the literature as K. pneumonia (see, for example, Streicher et al., Proc. Natl. Acad. Sci. 68:1174-1177 (1971 )).
  • the yiaX2 deletion mutant was constructed by joining sequences immediately upstream and downstream of the yiaX2 gene in a three-way ligation with the pMAK705 integration vector (described in Hamilton et al., J. Bacteriol. 171:4617-4622 (1989 )).
  • a fragment of about 1 kb in the orf1 gene was amplified using oligonucleotides 5'-ACCCAAGCTTCACCAAAAGAGTGAAGAGGAAG-3' (SEQ ID NO:17) and 5'-CGTATCTAGAAAAATATTCTGGTGATGAAGGTGA-3 (SEQ ID NO:18), and digested with HindIII and XbaI.
  • a fragment of a similar size in the lyxK gene was amplified with oligonucleotides 5'-AGACTCTAGATCCACATAAACGCACTGCGTAAAC-3' (SEQ ID NO:19) and 5'-GAGGGGATCCTGGCTTCGTGAACGATATACTGG-3' (SEQ ID NO:20), and digested with XbaI and BamHI.
  • the two resulting fragments were ligated together between the HindIII and BamHI sites of the vector pMAK705.
  • the resulting plasmid was transformed into K . oxytoca strain M5a1, and candidates in which the deletion construct had integrated by double crossover were obtained as described in Hamilton et al., supra (1989).
  • the designation of the resulting K. oxytoca ⁇ yiaX2 strain is MGK002.
  • the yiaX2 -deficient phenotype was verified by by PCR analysis.
  • the K. oxytoca ⁇ yiaX2 [ tkr idnO ] strain was determined to grow very inefficiently on 2,5-DKG as the sole carbon source, and not to grow on 2-KLG. Confirmation that yiaX2 encoded a polypeptide having 2,5-DKG and 2-KLG permease activities was obtained by determining that adding back the gene restored the ability of the K. oxytoca ⁇ yiaX2 [ tkr idnO ] to grow well on either 2,5-DKG or 2-KLG (see below).
  • a tester strain for the metabolic selection of nucleic acid molecules encoding 2,5-DKG permeases was prepared by engineering K. oxytoca ⁇ yiaX2 to express enzymes involved in the catabolism of 2,5-DKG to gluconic acid, which can be converted to carbon and energy. Enzymes capable of catabolizing 2,5-DKG to gluconic acid are encoded by the tkr and idnO genes of the tkr idnD idnO operon designated SEQ ID NO:13.
  • the tkr idnD idnO operon (SEQ ID NO:13) was subcloned into the high copy number vector pUC19 and the resulting clone, designated pDF33, was transformed into K. oxytoca ⁇ yiaX2 .
  • the resultingtester strain (designated MGK002[pDF33] or K. oxytoca ⁇ yiaX2 [ tkr idnO ]) thus expresses all polypeptides required for the utilization of 2,5-DKG as a sole carbon source, but is deficient in 2,5-DKG permease activity to transport extracellular 2,5-DKG into the cell. Therefore, a nucleic acid molecule that encodes a 2,5-DKG permease, upon expression in the tester strain, should confer the ability of the tester strain to grow on 2,5-DKG.
  • the metabolic selection strategy is shown schematically in Figure 3 .
  • the yiaX2 open reading frame (nucleotides 3777 to 5278 of SEQ ID NO:19 of WO 00/22170 ) was PCR-amplified using olignucleotides 5'-AATAGGATCCTTCATCACCAGAATATTTTTA-3' (SEQ ID NO:21) and 5'-CATAGGTACCGGCTTTCAGATAGGTGCC-3' (SEQ ID NO:22) digested with BamH1 and Kpn1 and ligated into pCL1920 (Lerner et al., Nucl.
  • K. oxytoca ⁇ yiaX2 [ tkr idnO ] transformed with the resulting construct, was able to grow overnight at 30°C on M9 minimal agar medium supplemented with either 2-KLG or 2,5-DKG (0.25%) and 0.1 mM IPTG. Therefore, K. oxytoca ⁇ yiaX2 [ tkr idnO ] was confirmed to be an appropriate tester strain to identify additional novel 2,5-DKG permeases, and to determine their selectivity.
  • the cloning vector used for constructing the above positive control and for preparing bacterial genomic libraries is plasmid pCL1920 (Lerner et al., supra , 1990), a low-copy number expression vector which carries a spectinomycin/streptomycin resistance determinant. Expression is driven by the lacPO promoter/operator region which is repressed by the lacI q gene product when provided by the host, and induced in the presence of 0.01 to 1mM IPTG.
  • Genomic DNA from the following species and isolates was prepared according to the method outlined below: Pantoea citrea (ATCC 39140), Klebsiella oxytoca MGK002 ( ⁇ yiaX2 ) , Pseudomonas aeruginosa, and a mixture of 25 environmental isolates, obtained from 18 different soil and water samples, and able to grow on 2,5-DKG as the sole carbon source.
  • Klebsiella oxytoca MGK002 ( ⁇ yiaX2 ) was among the bacteria chosen because there was a slight amount of background growth observable in the tester strain on 2,5-DKG as the sole carbon source, and some 2,5-DKG permease activity in an uptake assay.
  • the tester strain did not grow on 2-KLG, and exhibited no detectable 2-KLG uptake, suggesting the presence of a second 2,5-DKG permease with selectivity for 2,5-DKG in K. oxytoca .
  • genomic DNA was subjected to Sau3A controlled digestion in order to obtain fragments ranging between 3 to 20kb in size. Half that amount was ligated with the low-copy number expression vector pCL1920, which had previously been digested with BamHI and dephosphorylated.
  • the resulting genomic libraries were transformed into E.coli DH10B electrocompetent cells (GIBCO-BRL) and briefly amplified overnight at 30°C on LB-agar supplemented with 100 ⁇ g/ml spectinomycin.
  • 30,000 to 120,000 clones were plated out and plasmid DNA was bulk-extracted using standard procedures. Insert size was randomly checked and the amplified libraries were stored in the form of plasmid DNA at -20°C for further use in the tester strain.
  • MGK002 oxytoca ⁇ yiaX2 devoid of plasmid pDF33, to verify that the tkr idnD0 pathway was indeed required for growth of those clones on 2,5-DKG.
  • a brief genetic characterization was performed to eliminate identical clones. Following preliminary 2,5-DKG/ 2-KLG uptake assays, 5 clones were retained for further analysis: 2 originated from the Pantoea citrea library, 1 from K. oxytoca and 2 from the mixed environmental library.
  • DNA sequencing of the vector inserts revealed the presence of a nucleotide sequence (SEQ ID NOS:1, 3, 5, 7 and 9) encoding a polypeptide (SEQ ID NOS:2, 4, 6, 8 and 10) displaying homology with published transporters and with yiaX2 (SEQ ID NO:11, and its encoded polypeptide SEQ ID NO:12). Also present on these inserts were other orfs, and in most cases an endogenous promoter.
  • the insert containing both of the prmA and prmB orfs was about 9kb, and also contained an orf homologous to bacterial idnO , two orfs encoding transcriptional repressors, an orf of unknown function, and 3 orfs encoding homologs of E. coli polypeptides involved in nitrate utilization.
  • the insert containing the PE1 orf was about 3kb, and also contained a putative dehydro-deoxygluconokinase gene closely related to the B. subtilis kdgK gene and a homolog of the E. coli ydcG gene.
  • the insert containing the PE6 orf was about 6.7kb.
  • the genomic environment of PE6 appeared similar to the yia operon of E. coli and K . oxytoca, as SEQ ID NO:4 was preceded by a yiaL homolog and a yiaK homolog was also present on the insert.
  • the insert containing the PK1 orf was about 5.5kb. In contrast to the other inserts, this insert did not appear to contain an endogenous promoter, indicating that the PK1 orf was apparently transcribed from the vector's promoter. The PK1 orf was directly followed by a tkr homolog.
  • Nucleic acid molecules encoding each 2,5-DKG permease were reintroduced into K. oxytoca ⁇ yiaX2 [ tkr idnO ] and the resulting strains assayed for growth on 2,5-DKG and 2-KLG, and also assayed for uptake of radiolabeled 2,5-DKG and 2-KLG.
  • the uptake assays were performed by mixing radioactive 2-KLG or 2,5-DKG with IPTG-induced cells, removing aliquots at regular intervals, and measuring both the decrease in radioactivity in the supernatant and the appearance of radioactivity in the cells over time.
  • the results of the growth assays and 2,5-DKG uptake assay are shown in Table 1, below.
  • Nucleic acid molecules encoding the different permeases were also subcloned into a variety of vectors, including the high copy number vector pSE380 (which contains a tac promoter), the medium copy number vector pACYC184 (which is promoterless), or the low copy number vector pCL1920, and introduced into a Pantoea strain suitable for bioproduction of 2-KLG from glucose (see U.S. Patent No. 5,032,514 ). The resulting strains were assessed under biofermentation conditions to determine which combinations of nucleic acid molecules, promoters and vectors are optimal for enhancing 2-KLG production.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Transition And Organic Metals Composition Catalysts For Addition Polymerization (AREA)
  • Laminated Bodies (AREA)
  • Production Of Multi-Layered Print Wiring Board (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (27)

  1. Molécule d'acide nucléique isolé codant pour un polypeptide qui a une activité de 2,5-DKG perméase, ladite molécule d'acide nucléique comprenant
    (a) une séquence de nucléotides ayant au moins 80% d'identité avec une séquence de nucléotides sélectionnée dans le groupe consistant en SEQ ID NOS:1, 3, 5, 7 et 9; ou
    (b) une séquence de nucléotides sélectionnée dans le groupe consistant en SEQ ID NOS:1, 3, 5, 7 et 9; ou
    (c) une séquence de nucléotides qui code pour un polypeptide ayant au moins 80% d'identité avec une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NOS: 2, 4, 6, 8 et 10.
  2. Molécule d'acide nucléique isolé selon la revendication 1, qui code pour un polypeptide ayant une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NO:2, 4, 6, 8 et 10.
  3. Molécule d'acide nucléique isolé selon la revendication 1, fonctionnellement liée à un promoteur d'une expression de gènes.
  4. Molécule d'acide nucléique isolé selon la revendication 3, où ledit promoteur est un promoteur lac.
  5. Vecteur comprenant la molécule d'acide nucléique isolé selon la revendication 3 ou la revendication 4.
  6. Vecteur selon la revendication 5, comprenant un gène de résistance à la spectinomycine.
  7. Cellule bactérienne comprenant le vecteur de la revendication 5 ou de la revendication 6.
  8. Cellule bactérienne selon la revendication 7, où ladite molécule d'acide nucléique isolé comprend une séquence de nucléotides qui code pour un polypeptide qui a une activité de 2,5-DKG perméase, ledit polypeptide ayant une séquence d'acides aminés au moins à 80% identique à une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NOs:2, 4, 6, 8 et 10.
  9. Cellule bactérienne selon la revendication 8, où ladite séquence d'acides aminés est au moins à 95% identique à la SEQ ID NO:8.
  10. Cellule bactérienne selon la revendication 9, comprenant en outre:
    (a) une molécule d'acide nucléique isolé comprenant une séquence de nucléotides qui code pour un polypeptide ayant une séquence d'acides aminés au moins à 95% identique à la SEQ ID NO:4; ou
    (b) une molécule d'acide nucléique isolé comprenant une séquence de nucléotides qui code pour un polypeptide ayant une séquence d'acides aminés au moins à 95% identique à la SEQ ID NO:10.
  11. Cellule bactérienne selon la revendication 7,
    (a) qui est du gène Klebsiella; ou
    (b) qui est déficiente dans l'activité de 2,5-DKG endogène.
  12. Cellule bactérienne selon la revendication 11 (b), comprenant une molécule d'acide nucléique isolé codant pour un polypeptide ayant au moins 80% d'identité avec la SEQ ID NO:14 et une activité de 2-céto réductase.
  13. Cellule bactérienne selon la revendication 11(b), comprenant une molécule d'acide nucléique isolé codant pour un polypeptide ayant au moins 80% d'identité avec la SEQ ID NO: 16 et une activité de 5-céto réductase.
  14. Cellule bactérienne selon la revendication 7,
    (a) qui est du gène Pantoea; ou
    (b) qui exprime une enzyme qui catalyse la conversion de 2,5-DKG en 2-KLG.
  15. Cellule bactérienne selon la revendication 14(b), qui exprime des enzymes qui catalysent la conversion du glucose en 2,5-DKG.
  16. Cellule bactérienne selon la revendication 15, qui est déficiente dans l'activité d'endogène 2-céto-réductase.
  17. Méthode de réalisation de la molécule d'acide nucléique isolé selon la revendication 1, comprenant l'introduction dans une cellule bactérienne ayant une déficience en activité perméase endogène 2,5-DKG d'une ou de plusieurs molécules d'acide nucléique apte à être exprimées, identifier une cellule ayant une activité perméase 2,5-DKG à la suite de ladite introduction et isoler la molécule d'acide nucléique introduite de ladite cellule.
  18. Méthode selon la revendication 17, dans laquelle un ou plusieurs molécules d'acide nucléique isolé est une biobliothèque d'ADN génomique.
  19. Méthode selon la revendication 18, dans laquelle ladite bibliothèque d'ADN génomique est préparée à partir d'un échantillon environnemental.
  20. Méthode selon la revendication 17, dans laquelle ladite cellule bactérienne est une Klebsiella oxytoca déficiente en yiaX2.
  21. Méthode selon la revendication 17, où ladite cellule bactérienne comprend une molécule d'acide nucléique isolé codant pour un polypeptide ayant au moins 80% d'identité avec la SEQ ID NO:14 et une activité de 2-céto réductase, et un polypeptide ayant au moins 80% d'identité avec la SEQ ID NO:16 et une activité de 5-céto réductase.
  22. Méthode d'utilisation de la molécule d'acide nucléique isolé selon la revendication 1 pour augmenter la production de 2-KLG, comprenant l'expression du polypeptide codé par ladite molécule d'acide nucléique dans une cellule bactérienne qui exprime une enzyme qui catalyse la conversion de 2,5-DKG en 2-KLG, où la cellule bactérienne exprime en outre des enzymes qui catalysent la conversion de glucose en 2,5-DKG.
  23. Méthode selon la revendication 22, où ladite cellule bactérienne est déficiente en activité endogène 2-céto-réductase.
  24. Méthode selon la revendication 22, où ladite cellule bactérienne est du gène Pantoea.
  25. Méthode selon la revendication 22, comprenant en outre la conversion de 2-KGL en acide ascorbique.
  26. Polypeptide isolé qui a une activité perméase 2,5-DKG, ledit polypeptide comprenant:
    (a) une séquence d'acides aminés ayant au moins 80% d'identité avec une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NOS: 2, 4, 6, 8 et 10; ou
    (b) une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NOS: 2, 4, 6, 8 et 10.
  27. Anticorps spécifique pour un polypeptide isolé qui a une activité perméase 2,5-DKG, ledit polypeptide comprenant une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NOS: 2, 4, 6, 8 et 10.
EP01959520A 2000-08-04 2001-08-03 Permeases 2,5-dkg Expired - Lifetime EP1305422B1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US63329400A 2000-08-04 2000-08-04
US633294 2000-08-04
US67703200A 2000-09-29 2000-09-29
US677032 2000-09-29
PCT/US2001/024507 WO2002012468A2 (fr) 2000-08-04 2001-08-03 Permeases 2,5-dkg

Publications (2)

Publication Number Publication Date
EP1305422A2 EP1305422A2 (fr) 2003-05-02
EP1305422B1 true EP1305422B1 (fr) 2011-05-18

Family

ID=27091840

Family Applications (3)

Application Number Title Priority Date Filing Date
EP01961906A Withdrawn EP1305423A2 (fr) 2000-08-04 2001-08-03 Renforcement de production industrielle par augmentation du transport substratique
EP01959465A Expired - Lifetime EP1305421B1 (fr) 2000-08-04 2001-08-03 Production amelioree d'acide 2-ceto-l-gulonique
EP01959520A Expired - Lifetime EP1305422B1 (fr) 2000-08-04 2001-08-03 Permeases 2,5-dkg

Family Applications Before (2)

Application Number Title Priority Date Filing Date
EP01961906A Withdrawn EP1305423A2 (fr) 2000-08-04 2001-08-03 Renforcement de production industrielle par augmentation du transport substratique
EP01959465A Expired - Lifetime EP1305421B1 (fr) 2000-08-04 2001-08-03 Production amelioree d'acide 2-ceto-l-gulonique

Country Status (12)

Country Link
US (1) US20040029234A1 (fr)
EP (3) EP1305423A2 (fr)
JP (3) JP4975938B2 (fr)
CN (3) CN1469927A (fr)
AT (2) ATE318907T1 (fr)
AU (3) AU2001283132A1 (fr)
BR (3) BR0113036A (fr)
CA (4) CA2820132A1 (fr)
DE (1) DE60117566T2 (fr)
DK (1) DK1305421T3 (fr)
MX (3) MXPA03001026A (fr)
WO (3) WO2002012468A2 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6720168B2 (en) 2000-08-04 2004-04-13 Genencor International, Inc. 2,5-DKG permeases
CA2820132A1 (fr) * 2000-08-04 2002-02-14 Genencor International, Inc. Permeases 2,5-dkg
US7229811B2 (en) 2001-08-03 2007-06-12 Genencor International, Inc. 2,5-diketo-D-gluconic acid (2,5-DKG) permeases
JP2007525186A (ja) 2003-05-22 2007-09-06 ジェネンコー・インターナショナル・インク 非機能化された内因性グルコン酸トランスポーターを有する代謝を修飾したバクテリア系統
JP4742521B2 (ja) * 2003-06-05 2011-08-10 味の素株式会社 目的物質の製造法
US7335496B2 (en) 2003-06-05 2008-02-26 Ajinomoto Co., Inc. Method for producing target substance
WO2006084725A1 (fr) * 2005-02-11 2006-08-17 Dsm Ip Assets B.V. Nouveau gene sts 16
JP4980651B2 (ja) * 2006-06-09 2012-07-18 Ｊｘ日鉱日石エネルギー株式会社 菌体温度を制御する能力を有する細菌
CA2914003C (fr) * 2013-06-05 2018-01-02 Lanzatech New Zealand Limited Micro-organismes recombines presentant un flux accru par une voie de fermentation
CN114045243B (zh) * 2021-12-03 2023-12-26 山东天力药业有限公司 一种缩短生黑葡萄糖酸杆菌发酵周期的方法

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4757012A (en) * 1983-06-28 1988-07-12 Genentech, Inc. Ascorbic acid intermediates and process enzymes
US5047340A (en) * 1985-06-05 1991-09-10 The University Of Kentucky Research Foundation LAC+ saccharomyces cerevisiae
US5032514A (en) * 1988-08-08 1991-07-16 Genentech, Inc. Metabolic pathway engineering to increase production of ascorbic acid intermediates
US5376544A (en) * 1992-09-08 1994-12-27 Rutgers The State University Of New Jersey Enzymes for the production of 2-keto-L-gulonic acid
AU7854694A (en) * 1993-10-08 1995-05-04 Lonza A.G. Genes for butyrobetaine/crotonobetaine-l-carnitine metabolism and their use for the microbiological production of l-carine
US5795716A (en) * 1994-10-21 1998-08-18 Chee; Mark S. Computer-aided visualization and analysis system for sequence evaluation
DE19523279A1 (de) * 1995-06-27 1997-01-09 Forschungszentrum Juelich Gmbh Verfahren zur mikrobiellen Herstellung von Aminosäuren mittels rekombinanter Mikroorganismen mit erhöhter Sekretionsrate
FR2736652B1 (fr) * 1995-07-13 1997-08-14 Univ Bourgogne Levures et bacteries transformees pour operer la fermentation malolactique dans les vins
US5958672A (en) * 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US5795761A (en) * 1996-01-11 1998-08-18 Rutgers, The State University Of New Jersey Mutants of 2,5-diketo-D-gluconic acid (2,5-DKG) reductase A
US5783431A (en) * 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
US5817490A (en) * 1996-05-17 1998-10-06 Eastman Chemical Company Enzymatic process for the manufacture of ascorbic acid 2-keto-L-gulonic acid and esters of 2-keto-L-gulonic acid
CN1180747A (zh) * 1996-10-18 1998-05-06 中国科学院上海生物工程研究中心 从葡萄糖发酵制备2－酮基－l－古龙酸的新方法
US5834231A (en) * 1996-10-24 1998-11-10 Archer Daniels Midland Co. Bacterial strains and use thereof in fermentation process for 2-keto-L-gulonic acid production
DE19644566A1 (de) * 1996-10-26 1998-04-30 Forschungszentrum Juelich Gmbh Mikrobielle Herstellung von Substanzen aus dem aromatischen Stoffwechsel / I
EP0839909A1 (fr) * 1996-10-29 1998-05-06 Rijksuniversiteit te Groningen Séquences d'acides nucléiques codant pour des protéines transporteuses de citrate
CN1119414C (zh) * 1997-12-30 2003-08-27 中国科学院上海生物工程研究中心 突变的2,5二酮基－d－葡萄糖酸还原酶及其基因工程表达
US6368793B1 (en) * 1998-10-14 2002-04-09 Microgenomics, Inc. Metabolic selection methods
US6720168B2 (en) * 2000-08-04 2004-04-13 Genencor International, Inc. 2,5-DKG permeases
CA2820132A1 (fr) * 2000-08-04 2002-02-14 Genencor International, Inc. Permeases 2,5-dkg

Also Published As

Publication number Publication date
CN100378222C (zh) 2008-04-02
MXPA03001025A (es) 2003-11-25
JP2004519214A (ja) 2004-07-02
ATE510017T1 (de) 2011-06-15
CN1458976A (zh) 2003-11-26
WO2002012468A2 (fr) 2002-02-14
AU2001283132A1 (en) 2002-02-18
JP2004514421A (ja) 2004-05-20
CN100547073C (zh) 2009-10-07
WO2002012481A2 (fr) 2002-02-14
WO2002012481A3 (fr) 2002-10-10
EP1305423A2 (fr) 2003-05-02
JP4975937B2 (ja) 2012-07-11
AU2001281068A1 (en) 2002-02-18
BR0113036A (pt) 2006-02-21
BR0113034A (pt) 2005-08-16
EP1305421A2 (fr) 2003-05-02
CA2417970C (fr) 2013-09-24
US20040029234A1 (en) 2004-02-12
WO2002012468A3 (fr) 2002-12-05
MXPA03001030A (es) 2003-11-25
JP4975938B2 (ja) 2012-07-11
DK1305421T3 (da) 2006-07-10
EP1305421B1 (fr) 2006-03-01
CA2417871C (fr) 2011-11-15
WO2002012528A2 (fr) 2002-02-14
DE60117566D1 (de) 2006-04-27
EP1305422A2 (fr) 2003-05-02
ATE318907T1 (de) 2006-03-15
BR0113032A (pt) 2006-02-21
DE60117566T2 (de) 2007-01-11
CA2417871A1 (fr) 2002-02-14
CN1469927A (zh) 2004-01-21
CN1527880A (zh) 2004-09-08
MXPA03001026A (es) 2003-09-22
WO2002012528A3 (fr) 2003-02-06
CA2417970A1 (fr) 2002-02-14
BR0113034B1 (pt) 2014-09-16
AU2001281018A1 (en) 2002-02-18
JP2004519213A (ja) 2004-07-02
CA2417969A1 (fr) 2002-02-14
CA2820132A1 (fr) 2002-02-14

Similar Documents

Publication Publication Date Title
EP2348113B1 (fr) Production microbienne d'acide ascorbique L
EP1305422B1 (fr) Permeases 2,5-dkg
CA2597499A1 (fr) Micro-organismes recombinants dans la production par fermentation de vitamine c et un procede associe
US7229811B2 (en) 2,5-diketo-D-gluconic acid (2,5-DKG) permeases
US7229795B2 (en) 2,5-DKG permeases
US9079951B2 (en) Method for production of vitamin C
US8053218B2 (en) Gene STS 18
WO2006084703A2 (fr) Nouveau gene sts 24
WO2006084701A1 (fr) Gene phosphotransferase a partir de gluconobacter oxydans
US20090215135A1 (en) Novel Gene STS 22
WO2006084704A2 (fr) Nouveau gene sts 01
WO2006084702A2 (fr) Nouveau gene sts 23
WO2006084725A1 (fr) Nouveau gene sts 16
WO2006084720A2 (fr) Nouveau gene sts 25
WO2006084705A2 (fr) Nouveau gene sts 06
WO2006084722A2 (fr) Nouveau gene sts 15
WO2006084716A1 (fr) Gene sms 14
WO2009087221A1 (fr) Nouveau gène sms 43
WO2006084729A1 (fr) Gene rcs o1
EP1859030A1 (fr) Gene sms 05
WO2006084749A2 (fr) Gene rcs 02

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030224

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

R17C First examination report despatched (corrected)

Effective date: 20061116

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 15/31 20060101AFI20101117BHEP

Ipc: C07K 14/24 20060101ALI20101117BHEP

Ipc: C07K 14/26 20060101ALI20101117BHEP

Ipc: C12P 7/60 20060101ALI20101117BHEP

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MICROGENOMICS, INC.

Owner name: DANISCO US INC.

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 60144655

Country of ref document: DE

Effective date: 20110630

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20110518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110919

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110819

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110829

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110831

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

26N No opposition filed

Effective date: 20120221

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110831

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110831

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 60144655

Country of ref document: DE

Effective date: 20120221

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110803

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20110803

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110518

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20140730

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20140808

Year of fee payment: 14

Ref country code: GB

Payment date: 20140730

Year of fee payment: 14

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 60144655

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20150803

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20160429

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160301

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150803

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150831